Immunostimulatory oligonucleotides and uses thereof

ABSTRACT

Oligonucleotides containing the non-palindromic sequence motif:
 
X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 ,
 
wherein X 1  is C,T,G or A (preferably T or C); wherein X 2  is C,T,G or A; wherein X 7  is C,T,G or A (preferably G); at least three, and preferably all, of X 3 , X 4 , X 5 , X 6  and X 8  are T; and with the proviso that, in the motif, a C does not precede a G (in other terms, the nucleic acid motif does not consist of a CpG oligonucleotide), that modulate the immune response of animals of the order Primate, including humans, are disclosed. This immune modulation is characterized by stimulation of proliferation, differentiation, cytokine production and antibody production on B-cells and cell differentiation on plasmacytoid dendritic cells.

FIELD OF THE INVENTION

The present invention relates generally to oligonucleotides containingthe non-palindromic sequence motif:X₁X₂X₃X₄X₅X₆X₇X₈,wherein X₁ is C,T,G or A (preferably T or C); X₂ is C,T,G or A; X₇ isC,T,G or A (preferably G); at least three, and preferably all, of X₃,X₄, X₅, X₆ and X₈ are T; with the proviso that, in the motif, a C doesnot precede a G, that are immunostimulatory in animals of the orderPrimate, including humans.

RELEVANT REFERENCES Patent Documents

-   U.S. Pat. No. 5,663,153-   U.S. Pat. No. 5,723,335-   U.S. Pat. No. 6,090,791-   U.S. Pat. No. 6,194,388-   U.S. Pat. No. 6,207,646-   U.S. Pat. No. 6,239,116-   U.S. Pat. No. 6,429,199-   EP 0468520-   WO 95/26204-   WO 96/02555-   WO 98/40100-   WO 98/52962-   WO 99/33488-   WO 99/56755-   WO 00/62802-   WO 01/00231-   WO 01/00232-   WO 01/55370

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BACKGROUND OF THE INVENTION

The Immune System

The major function of the immune system is to protect the host ofinvading pathogens. A number of different cell types, bothantigen-independent and antigen-specific, have evolved to detect andneutralize these invading pathogens. Among them, lymphocytes have animportant characteristic, which is their ability to specificallyrecognize antigens, a feature not possessed by any other cell. Thismeans that any lymphocyte function stimulated by an antigen is directedsolely at that antigen.

Lymphocytes may be divided into two major populations: T and B. Tlymphocytes have a central role in regulating the immune response andfor this they produce and secrete lymphokines (i.e.: interleukins).B-lymphocytes are the only cells that produce antibodies, which areproteins—Immunoglobulins (IgG)—that recognize and bind antigens.

Some T lymphocytes are known as helper (Th lymphocytes) because theyassist B cells to produce antibody. T-lymphocytes express acharacteristic membrane molecule designated CD4. Other T lymphocytes areknown as cytotoxic (CTL) because they are capable of killing certaincells. They express a different characteristic membrane proteindesignated CD8.

Th lymphocytes, in mice, have been subdivided according to thelymphokines they produce in groups designated Th0, Th1 and Th2. Ingeneral, Th 1 lymphocytes produce lymphokines which stimulatemacrophages and CTLS (IL2, IFNγ, TNF-β), Th 2 lymphocytes producelymphokines which stimulate B lymphocytes to proliferate and produceantibody (IL 2, IL5, IL6, IL10, IL13), whilst Th 0 lymphocytes produce amixture of lymphokines and are thought to be an intermediate stage fromwhich Th 1 and Th 2 lymphocytes are derived. In humans, Th1 and Th2 likelymphocytes have been demonstrated, although they do seem to show a lessstrict division with respect to their patterns of cytokine secretion.

A third population of lymphocytes, which lack the major makers of T andB cells include the natural killer cells (NK cells), the killer cells (Kcells) and the lymphokine-activated killer cells (L A K cells). NK cellscan kill certain tumor cells and some virally infected cells, but unlikecytotoxic T lymphocytes they are not capable of recognizing a specificantigen. K cells are able to bind to cells, which have antibody to themvia their antigen-binding regions and kill them. L A K cells do notspecifically recognize an antigen but they are capable of destroying awider range of targets a NK cells.

Macrophages and dendritic cells play a critical role in initiatingimmune responses, helping T cells to respond to antigens.

There are several antibody classes. The IgG class comprises most of thecirculating antibodies and it has four subclasses designated IgG1, IgG2,IgG3 and IgG4.

The IgM class comprises about 10% of the circulating antibodies. Theseare the principal antibodies produce during the primary immunologicalresponse. The IgA class comprises most of the antibody secreted atmucous membranes and exerts its protective effect by blocking access ofthe antigen to the inner body. The IgD class comprises less than 1% ofserum antibodies and its biological role is largely unknown. The IgEclass comprises antibodies that are mainly bound to the surface of mostcells and basophils. These antibodies are associated with reactions thatoccur in individuals who are undergoing allergic reactions.

Vaccines and Vaccines Adjuvants

Vaccines are preparations used to stimulate animals to mount an immuneresponse against antigens included in the vaccine.

Vaccines often include adjuvants, which are substances that used incombination with specific antigen produce more immunity than the antigenused alone. (Ramon, G., 1926. Procedes pour accroite la production desantitoxins. Ann. Inst. Pasteur. 40, 1-10).

Many kind of compounds function as vaccine adjuvants (Edelman, R., 2000.An overview of adjuvant use, in: Vaccine Adjuvants. Preparation Methodsand Research Protocols. D. T. O'Hagan, Ed., Humana Press, Totowa, N.J.References cited in this article are incorporated herein as backgroundmaterial). However, currently, the only adjuvants approved for use inhumans are aluminum salts (Gupta, R. K. and Rost, B. E., 2000. Aluminumcompounds as vaccine adjuvants in: Vaccine Adjuvants. PreparationMethods and Research Protocols. D. T. O'Hagan, Ed., Humana Press,Totowa, N.J.) and the oil-in-water emulsion M F 59 (Ott, G.Radhakrishman, R. Fang, J. and Hora, M., 2000. The adjuvant M F 59: A10-Year Perspective, in : Vaccine Adjuvants. Preparation Methods andResearch Protocols. D. T. O'Hagan, Ed., Humana Press, Totowa, N.J.).

Nucleic Acids as Immunostimulatory Compounds

Several polynucleotides have been demonstrated to have immunostimulatoryproperties. For example, poly (I, C) is an inducer of interferon (IFN)production, macrophage activation and NK cell activation (Talmadge, J.E., Adams, J., Phillips, H., Collins, M., Lenz, B., Schneider, M.,Schlick, E., Ruffmann, R., Wiltrout, R. H., Chirigos, M. A. 1985.Immunomodulatory effects in mice of polyinosinic-polycytidylic acidcomplexed with poly-L:-lysine and carboxymethylcellulose. Cancer Res.45:1058; Wiltrout, R. H., Salup, R. R., Twilley, T. A., Talmage, J. E.1985. mmunomodulation of natural killer activity by polyribonucleotides.J. Biol. Resp. Mod. 4:512), poly (dG,dC) is mitogenic for B cells(Messina, J. P., Gilkerson, G. S., Pisetsky, D. S. 1993. The influenceof DNA structure on the in vitro stimulation of murine lymphocytes bynatural and synthetic polynucleotide antigens. Cell. Immunol. 147:148)and induces IFN and NK activity (Tocunaga, T., Yamamoto, S., Namba,K.1988. A synthetic single-stranded DNA, poly(dG,dC), inducesinterferon-α/β and -γ, augments natural killer activity, and suppressestumor growth. Jpn.J. Cancer Res. 79:682).

Bacterial DNA has also been reported to have immunostimulatoryproperties. These properties include the induction of cytokines(interferon gamma (IFN γ), alpha (IFN α), beta (IFN β); tumor necrosisfactor alpha (TNF α), interleukin 6 (IL6), 12 (IL 12) and 18 (IL 18), aswell as the direct stimulation of B cells (Yamamoto, S. et al. 1988. Invitro augmentation of natural killer cell activity of interferon α/β andγ with deoxyribonucleic acid fraction from Mycobacterium bovis BCG. Jpn.J. Cancer Res. (Gann) 79: 866-873; Yamamoto S. et al, 1992. DNA frombacteria, but not from vertebrates, induces interferons, activatesnatural killer cells and inhibits tumor growth. Microbiol. Immunol. 36:983-997.; Klinman, D. M., Yi, A-K., Beaucage, S. L., Conover, J. andKrieg, A. M., 1996.

CpG motifs present in bacterial DNA rapidly induce lymphocytes tosecrete interleukin 6, interleukin 12 and interferon γ. Proc. Natl.Acad. Sci. USA 93, 2879-2883. Halpern, M. D., et al. 1996. Bacterial DNAinduces murine interferon-γ production by stimulation of interleukin-12and tumor necrosis factor-α. Cell. Immunol. 167: 72-78. Sparwasser, T.et al, 1997. Macrophages sense pathogens via DNA motifs: induction oftumor necrosis factor-α-mediated shock. Eur. J. Immunol. 27: 1671-1679;Krieg, A. M. et al., 1995. CpG motifs in bacterial DNA trigger directB-cell activation. Nature 374: 345-349).

In contrast, it has been reported that mammalian DNA has no significantimmune effects (Pisetsky, D. S. 1996. The immunologic properties of DNA.J. Immunol. 156: 421-423; Messina et al. 1991. Stimulation of in vitromurine lymphocyte proliferation by bacterial DNA. J. Immunol. 147:1759).

Synthetic DNA has also been reported to be immunostimulatory if containsunmethylated CpG motifs. (Yamamoto, S et al.; 1992. Unique palindromicsequences in synthetic oligonucleotides are required to induce INF andaugment INF-mediated natural killer activity. J. Immunol. 148:4072-4076; Ballas, Z. K., et al.; 1996. Induction of NK activity inmurine and human cells by CpG motifs in oligodeoxynucleotides andbacterial DNA. J. Immunol. 157: 1840-1845; Hartmann, G., Krieg, A. M.2000. Mechanism and function of a newly identified CpG DNA motif inhuman primary B cells. J. Immunol. 164:944; Hartmann, G., Weeratna, R.D., Ballas, Z. K., Payette, P., Blackwell, S., Suparto, I., Rasmussen,W. L., Waldschmidt, M., Sajuthi, D., Purcell, R. H., Davis, H. L.,Krieg, A. M. 2000. Delineation of a CpG phosphorothioateoligodeoxynucleotide for activating primate immune responses in vitroand in vivo. J. Immunol. 164:1617; Verthelyi, D., Ishii, K. J., Gursel,M., Takeshita, F., Klinman, D. M. 2001. Human peripheric blood cellsdifferentially recognize and respond to two distinct CpG motifs. J.Immunol. 166:2372). However, one oligonucleotide containingphosphorothioate bonds that lack CpG motifs has been found to have someimmunostimulatory activity on human B cells (Liang, H., Nishioka, Y.,Reich, C. F., Pisetsky, D. S., Lipsky, P. E. 1996. Activation of human Bcells by phosphorothioate oligonucleotides. J. Clin. Invest. 98:1119).This particular non-CpG oligonucleotide containing phosphorothioatebonds is a poy-T chain, 20 nucleotides long. Also, Vollmer et al(Vollmer J, Janosch A, Laucht M, Ballas Z K, Schetter C, Krieg A M.Highly immunostimulatory CpG-free oligodeoxynucleotides for activationof human leukocytes. Antisense Nucleic Acid Drug Dev. 12:165-175, 2002)reported immunostimulation by phosphorothioate poly T ODNs. Theseauthors pointed out that poly T ODNs are only active as phosphorothioateODNs and have much lower activity than CpG ODNs.

It has now been discovered that non-CpG oligonucleotides containing thefollowing non-palindromic sequence motif:X₁X₂X₃X₄X₅X₆X₇X₈,wherein X₁ is C,T,G or A (preferably T or C); X₂ is C,T,G or A; X₇ isC,T,G or A (preferably G); at least three, and preferably all, of X₃,X₄, X₅, X₆ and X₈ are T; with the proviso that, in the motif, a C doesnot precede a G, have potent immunostimulatory activity. Therefore,these oligonucleotides can be administered to subjets to treat “immunesystem deficiencies” or in conjunction with a vaccine, as adjuvants, toboost the immune system in order to have a better response to thevaccine or administered to subjects to increase the responsiveness totumors.

SUMMARY OF THE INVENTION

It has now been discovered that non-CpG oligonucleotides containing thefollowing non-palindromic sequence motif:X₁X₂X₃X₄X₅X₆X₇X₈,wherein X₁, X₂ and X₇ are C,T,G or A; at least three of X₃, X₄, X₅, X₆and X₈ are T; with the proviso that, in the motif, a C does not precedea G, are useful as inmmunostimulants in animals of the order Primate,including humans.

According to a preferred embodiment, X₁ consist of a C or a T and X₇consist of a G. More preferably X₃,X₄,X₅,X₆ X₇ X₈ of theimmunostimulatory motif consist of TTTTGT. Even more advantageouslyX₁X₂X₃X₄X₅X₆X₇X₈ consist of CNTTTTGT or TNTTTTGT wherein N is C, T, G orA. Those of X₃-X₆ and X₈ that are not T can be any nucleotide (e.g., C,T, G, A) or can be absent so that the nucleotide preceding linksdirectly with the nucleotide following the position of the omittednucleotide (S). The oligonucleotides of this invention are useful asadjuvants in a vaccine formulation comprising one or more antigens. Inembodiments of this aspect, the vaccine formulation can be liquid orlyophilized in dosage form. Many dosage forms are known in the art andcan be applied herein. In embodiments of this aspect theoligonucleotides of this invention are present in the composition at adose of from about 10 to 10,000 μg per dose. In these preparations theoligonucleotides of this invention may be combined with otherimmunostimulant compounds. Examples of well known immunostimulants are:α-interferon, β-interferon, γ-interferon, granulocyte macrophage colonystimulator factor (GM-CSF), interleukin 2 (IL2), interleukin 12 (IL12)and CpG oligonucleotide.

In preferred embodiments the antigenic component of the vaccine is oneor more, natural or recombinant, antigens of viruses like: Humanimmunodeficiency viruses, such as HIV-1 and HIV-2, polio viruses,hepatitis A virus, human coxsackie viruses, rhinoviruses, echoviruses,equine encephalitis viruses, rubella viruses, dengue viruses,encephalitis viruses, yellow fever viruses, coronaviruses), vesicularstomatitis viruses, rabies viruses, ebola viruses, parainfluenzaviruses, mumps virus, measles virus, respiratory syncytial virus,influenza viruses, Hantaan viruses, bunga viruses, hemorrhagic feverviruses, reoviruses, orbiviuises, rotaviruses, Hepatitis B virus,parvoviruses, papilloma viruses, polyoma viruses, adenoviruses), herpessimplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus(CMV), variola viruses, vaccinia viruses, pox viruses, African swinefever virus, the unclassified agent of delta hepatitis, the agents ofnon-A, non-B hepatitis; of infectious bacteria like: Helicobacterpylori, Borrelia burgdorferi, Legionella pneumophila, Mycobacteriumtuberculosis, Mycobacterium bovis (BCG), Mycobacterium avium,Mycobacterium intracellulare, Staphylococcus aureus, Neisseriagonorrhoeae, Neisseria meningitidis, Listeria monocytogenes,Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilusinfluenzae, Moraxella catharralis, Klebsiella pneumoniae, Bacillusanthracis, Corynebacterium diphtheriae, Clostridium perfringers,Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae,Pasturella multocida, and Treponema pallidum; of infectious fungi like:Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis,Blastomyces dermatitidis, Candida albicans; of infectious protists like:Plasmodium falciparum, Trypanosoma cruzi, Leishmania donovani andToxoplasma gondii. and; of human tumoral cells.

In embodiments of this aspect one or more of the oligonucleotides ofthis invention and the antigen are administered simultaneously locally(by oral, rectal, intranasal or transdermal route) or systemically (byintradermic or intramuscular injection). An aspect of this invention isa method of vaccination of a person. The person can be vaccinatedprophylactically or therapeutically.

A prophylactic vaccine is designed to elicit protection from a diseasecaused by an infectious agent through the induction of specificimmunity.

A therapeutic vaccine is designed to induce remission of an illness(i.e. a tumor and metastasis or illness associated with, an infectiousagent like the human immunodeficiency virus).

The method of vaccination includes administering one or more of theoligonucleotides of this invention and one or more antigens—that is, thevaccine can be designed against one disease target or a combination ofdisease targets.

Another aspect of this invention is a method of treatment of a personwith a tumoral disease or an immunological disorder, with one or more ofthe oligonucleotides of this invention to stimulate his/her endogenousimmune response. Examples of tumoral disease are: Chronic MyelogenousLeukemia, Precursor B-lymphoblastic lymphoma, B-cell chronic lymphocyticleukaemia, Lymphoplasmacytic lymphoma, Mantle cell lymphoma, Folliclecenter lymphoma, (follicular and diffuse), Marginal zone-B lymphoma,Extranodal lymphoma, Nodal marginal zone B-cell lymphoma, Splenicmarginal zone B-cell lymphoma, Hairy cell leukaemia, Plasmocytoma,Diffuse large B-cell lymphoma, Burkitt's lymphoma, High grade B-celllymphoma, Burkitt like, Melanoma, Kaposi's Sarcoma, Multiple Myeloma,Renal Cell Carcinoma, Bladder Cancer, Lung Cancer, Skin Cancer, BreastCancer, Colon Cancer and Uterus Cancer. Examples of immunologicaldisorders are: Allergy, Severe Combined Immunodeficiency, ChronicGranulomatous disease, and Acquired Immunodeficiency Disease.

In embodiments of this aspect, one or more of the oligonucleotide ofthis invention is/are present in a pharmaceutical formulation that canbe liquid or lyophilized in dosage form. Many dosage forms are known inthe art and can be applied herein. In embodiments of this aspect one ormore of the oligonucleotides of this invention is/are present in thecomposition at a dose of from about 10 to 10,000 μg per dose. In thesepreparations one or more of the oligonucleotides of this invention maybe combined with other immunostimulant compounds. Examples of well knownimmunostimulants are: α-interferon, β-interferon, γ-interferon,granulocyte macrophage colony stimulator factor (GM-CSF), interleukin 2(IL2), interleukin 12 (IL12), CpG oligonucleotides and Mycobacteriumbovis BCG cells. Also, one or more of the oligonucleotides of thisinvention may be combined with an antiinfective or anticancer drug, or asurgical procedure. In all these cases the oligonucleotides of thisinvention may be administered before, after or simultaneously with thealternative treatment.

Another aspect of this invention is a method of treatment of a personwith a tumoral disease or an immunological disorder, contactinglymphocytes or plasmacytoid dendritic cells from the subject with one ormore of the oligonucleotides of this invention “ex vivo” andreadministering the activated cells to the subject. In embodiments ofthis aspect one or more of the oligonucleotides of this invention arepresent in the incubation media in a concentration of about 0.10 to 100μg per ml.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph plotting the proliferation index of human peripheralblood mononuclear leukocytes (PBMC) cultured with the phosphorothioateODN IMT 023 (Seq. ID No 9), the phosphorothioate CpG ODN 2006:

5′ TCGTCGTTTTGTCGTTTTGTCGTT 3′,

the phosphorothioate non-CpG ODN IMT 021 (SEQ ID NO: 1). Data representthe mean and standard deviation of five independent assays.

FIG. 2 shows the results from a flow cytometry study using humanperipheral blood mononuclear leukocytes (PBMC) to determine thecomparative effect of the phosphorothioate CpG ODN 2006:

5′ TCGTCGTTTTGTCGTTTTGTCGTT 3′

and the phosphorothioate non-CpG ODN IMT 021 (SEQ ID NO: 1)on activationof the B cell population:

(2a) Representative flow cytometry diagrams comparing B cell activationby IMT 023, 2006 and IMT 021 (2b) Representative histograms of the flowcytometry diagrams showed in (a).

FIG. 3 shows the influence of the position of the immunostimulatorysequence motif (3a) X₁X₂X₃X₄X₅X₆X₇X₈ here disclosed on theimmunostimulatory activity of non-CpG ODNs as measured by proliferationassay (3b) and IL-6 secretion assay (3c). Data represent the mean andstandard deviation of three independent assays performed byquadruplicate each.

FIG. 4 shows the induction of CD40 (4a), MHC I (4b) and MHC II (4c) onCD19+ cells (B cells) by non-CpG-ODNs bearing the immunostimulatorysequence motif X₁X₂X₃X₄X₅X₆X₇X₈ here disclosed. Human PMBC were culturedfor 24 hr with indicated ODNs and then stained with fluorescentanti-CD19/anti-CD40 (4a) or, anti-CD19/anti-MHC I (4b) or, anti-MHC II(4c). Flow cytometric results are presented as histograms. Openhistograms correspond to cells cultured in absence of ODN and shadedhistograms to cells cultured in presence of ODN. ODN (S) meansphosphorothioate ODN.

FIG. 5 shows the induction of CD86 (5a), CD40 (5b) and MHC I (5c) onpurified B cells by non-CpG-ODNs bearing the immunostimulatory sequencemotif X₁X₂X₃X₄X₅X₆X₇X₈ here disclosed. Human purified B cells werecultured for 24 hr with indicated ODNs and then stained with fluorescentanti-CD19/anti-CD86 (5a) or, anti-CD19/anti-CD40 (5b) or,anti-CD19/anti-MHC I (5c). Flow cytometric results are presented ashistograms. Open histograms correspond to cells cultured in absence ofODN and shaded histograms to cells cultured in presence of ODN. ODN (S)means phosphorothioate ODN.

FIG. 6 shows induction of CD86 (6a), CD40 (6b) and MHC I (6c) in CD19+cells (B cells) by phosphodiester non-CpG-ODNs bearing theimmunostimulatory sequence motif X₁X₂X₃X₄X₅X₆X₇X₈ here disclosed. HumanPMBC were cultured for 48 hr with indicated phosphodiester ODNs and thenstained with fluorescent anti-CD19/anti-CD86 (6a) or,anti-CD19/anti-CD40 (6b) or, anti-CD19/anti-MHC I (6c). Flow cytometricresults are presented as histograms. Open histograms correspond to cellscultured in absence of ODN and shaded histograms to cells cultured inpresence of ODN. ODN (O) means phosphodiester ODN.

FIG. 7 shows stimulation of PMBC proliferation by non-CpG ODNs bearingthe motif X₁X₂X₃X₄X₅X₆X₇X₈ here disclosed in non-human primates. Cebusapella (7a) or Macacca fascicularis PMBC (7b) were cultured for 72 hrwith indicated ODNs (6 μg/ml). Data represent the mean and standarddeviation of four replicates.

FIG. 8 shows the induction of CD86 (8a), CD40 (8b) and MHC I (8c) on CD19+ (B cells) of a patient suffering B cell leukemia by non-CpG-ODNsbearing the motif X₁X₂X₃X₄X₅X₆X₇X₈ here disclosed. PMBC were culturedfor 24 hr with indicated ODNs and then stained with fluorescentanti-CD19/anti-CD86 (8a) or, anti-CD19/anti-CD40 (8b) or,anti-CD19/anti-MHC I (8c). Flow cytometric results are presented ashistograms. Open histograms correspond to cells cultured in absence ofODN and shaded histograms to cells cultured in presence of ODN. ODN (S)means phosphorothioate ODN.

FIG. 9 shows the induction of CD86 (9a), CD40 (9b) and MHC I (9c) onpurified plasmacytoid dendritic cells by non-CpG-ODNs bearing the motifX₁X₂X₃X₄X₅X₆X₇X₈ here disclosed. More than 95% pure plasmacytoiddendritic cells were cultured for 24 hr with indicated ODNs and thenstained with fluorescent anti-CD19/anti-CD86 (9a) or,anti-CD19/anti-CD40 (9b) or, anti-CD19/anti-MHC I (9c). Flow cytometricresults are presented as histograms. Open histograms correspond to cellscultured in absence of ODN and shaded histograms to cells cultured inpresence of ODN. ODN (S) means phosphorothioate ODN.

DETAILED DESCRIPTION OF THE INVENTION

Definitions:

An “allergy” refers to acquired hypersensitivity to a substance(allergen). Examples of allergies are eczema, allergic rhinitis, asthmaand urticaria.

An “immune system deficiency” refers to a disease in which the immunesystem is not functioning in normal capacity.

As used herein, the term “oligonucleotide” or “oligo” shall meanmultiple nucleotides (i.e. molecules comprising a sugar (e.g. ribose ordeoxyribose) linked to a phosphate group and to an exchangeable organicbase, which is either a substituted pyrimidine (e.g. cytosine (C),thymine (T) or uracil (U)) or a substituted purine (e.g. adenine (A) orguanine (G)). The term “oligonucleotide” as used herein refers to botholigoribonucleotides (ORNs) and oligodeoxyribonucleotides (ODNs). Theterm “oligonucleotide” shall also include oligonucleosides (i.e. anoligonucleotide minus the phosphate) and any other organic basecontaining polymer. Oligonucleotides can be obtained from existingnucleic acid sources (e.g. genomic or cDNA), but are preferablysynthetic (e.g. produced by oligonucleotide synthesis)

An “oligonucleotide” refers to multiple nucleotides linked byphosphodiester bonds.

An “immunostimulatory oligonucleotide” refers to an oligonucleotidewhich stimulates, (i.e. has a mitogenic effect on, or induces orincreases cytokine expression by) a cell of the immune system (ie: alymphocyte, a macrophage).

A “CpG” refers to an unmethylated cytosine-guanine dinucleotide.

A “CpG oligonucleotide” refers to an oligonucleotide which stimulates acell of the immune system and its immunostimulatory activity criticallydepends of the presence of at least one CpG in its sequence.

A “non-CpG oligonucleotide” refers to an oligonucleotide whichstimulates a cell of the immune system and its immunostimulatoryactivity does not critically depends of the presence of a CpG in itssequence.

A “subject” refers to an animal of the order Primate, including humans.

As used herein, the term “treating” refers to a process by which thesymptoms of a disease, and more particularly infectious diseases ortumoral diseases or immunological disorders are ameliorated orcompletely eliminated.

As used herein, the term “preventing” refers to a process by which adisease, and more particularly infectious diseases or tumoral diseasesor immunological disorders are obstructed or delayed.

In a preferred embodiment, the immunostimulatory oligonucleotides of theinvention are advantageously modified into stabilized oligonucleotides.Such stabilized immunostimulatory oligonucleotide may be particularlyuseful to obtain a prolonged immunostimulation. As used herein, a“stabilized oligonucleotide” refers to an oligonucleotide that isrelatively resistant to in vivo degradation (e.g. via an exo- orendo-nuclease). Preferred stabilized oligonucleotides of the presentinvention comprise a phosphate backbone modification. More particularly,the phosphate backbone modification is preferably a 5′ inter-nucleotidelinkage modification, for instance, at the first two nucleotides of the5′ end of the oligonucleotide of the invention. Furthermore, thephosphate backbone modification may be a 3′ inter-nucleotide linkagemodification. In such a case, the modification may occur, for instance,at the last two nucleotides of the 3′ end of the oligonucleotide of theinvention. Even more preferably, the immunostimulatory oligonucleotideof the invention may be stably modified so as to comprise aphosphorothioate-linked nucleotide (i.e. at least one of the phosphateoxygens is replaced by sulfur). In the most preferred embodiment, mostif not all the nucleotides of the immunostimulatory oligonucleotides ofthe invention comprise a phosphorothioate-linked nucleotide.

Other stabilized oligonucleotides may alternatively include: nonionicDNA analogs, such as alkyl- and aryl-phosphonates (in which the chargedphosphonate oxygen is replaced by an alkyl or aryl group),phosphodiester and alkylphosphotriesters, in which the charged oxygenmoiety is alkylated. Oligonucleotides which contain a diol, such astetraethyleneglycol or hexaethyleneglycol, at either or both terminihave also been shown to be substantially resistant to nucleasedegradation.

Description:

The present invention provides methods to augment the immune response ofanimals of the order Primate, including humans, adding to vaccines oneor more of the oligonucleotides of this invention or performing atreatment based in the administration of one or more of theoligonucleotides of this invention to a person with a tumoral disease oran immunological disorder or contacting white blood cells obtained froma person with a tumoral disease or an immunological disorder with one ormore of the oligonucleotides of this invention “ex vivo”, andreadministering these activated white blood cells to the same person.

Vaccines compositions useful containing one or more of theoligonucleotides of this invention can present antigens directly (i.e.,in the form of a defined protein or polysaccharide) or as a part of acomplex biological entity (i.e. complete viruses; complete bacterialcells; bacterial membranes or artificial conjugates likepolysaccharide-protein conjugates). These antigens can be combined inmultiple vaccines.

A vaccine composition including at least one antigen is formulated toinclude one or more of the oligonucleotides of this invention.

For example the antigen can be Moraxella catharralis killed cells orsubcellular fractions of these cells or Hepatitis B virus surfaceantigen natural or produced by means of the DNA recombinant technology.

One or more of the oligonucleotides of this invention may be formulatedalone or together with one or more antigens in a pharmaceuticalcomposition, which may also include carriers, thickeners, diluents,buffers, preservatives, surface active agents, anti-microbial agents,anti-inflammatory agents, anesthetics and the like. The formulation canbe liquid or lyophilized. The pharmaceutical composition may beadministered in a number of ways depending of whether local or systemictreatment is desired, and on the area to be treated. Administration maybe done topically, orally, by inhalation or parenterally. Formulationfor topical administration may include ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Pharmaceuticalcarriers, aqueous, powder or oily bases, thickeners and the like may benecessary or desirable. Formulations for oral administration includepowders or granules, suspensions or solutions in water or non-aqueousmedia, capsules, tablets and the like. Thickeners, flavorings, diluents,emulsifiers and the like may be necessary or desirable. Formulations forparenteral administration include sterile aqueous solutions, which mayalso contain buffers, diluents and other additives. A vaccine containingone or more antigens and one or more of the oligonucleotides of thisinvention can be formulated and used for prophylactic or therapeuticpurposes.

Common antigens used in viral prophylactic vaccines are from Hepatitis Bvirus, Hepatitis A virus and Influenza virus. Common antigens used inbacterial prophylactic vaccines are from Streptococcus pneumoniae,Haemophilus influenzae, Moraxella catharralis, Klebsiella pneumoniae andMycobacterium bovis (BCG). Common antigens used in therapeutic vaccinesare from Papilloma virus, HIV virus and melanoma cells.

A further refinement of a vaccine formulation is to incorporate one ormore of the oligonucleotides of this invention as adjuvant/s and theantigen/s into a delivery vehicle to provide for delayed release of theactive compounds of the vaccine over time. This can be accomplished byvarious means known in the art. Examples of these means areencapsulation into Poly (lactide-coglicolide) micro particles (Kersten,G. F. A. and Gander, B. 1996. Biodegradable Micro Spheres as vehiclesfor antigens, in: S. H. E. Kaufmann, ed. Concepts in VaccineDevelopment. Walter de Gruyter. Berlin-N.Y.), liposomes (Gregoriadis, G.et al. 2000. Liposomes as Immunological Adjuvants and Vaccine Carriers,in: S. H. E. Kaufmann, ed. Concepts in Vaccine Development. Walter deGruyter. Berlin-N.Y.) and poly (methyl methacrylate) nanoparticles(Kreuter, J. 2000. Poly (Methyl Methacrylate) nanoparticles as vaccineadjuvants, in: S. H. E. Kaufmann, ed. Concepts in Vaccine Development.Walter de Gruyter. Berlin-N.Y.).

Another refinement of the vaccine formulation is to conjugate theantigen/s and one or more of the oligonucleotides of this invention, bychemical means (Mier W, Eritja R, Mohammed A, Haberkorn U, Eisenhut M.2000. Preparation and evaluation of tumor-targetingpeptide-oligonucleotide conjugates. Bioconjug. Chem. 11:855).

Many vaccine formulations are known in the art and can be used bysubstituting one or more of the oligonucleotides of this invention forthe adjuvant previously known or by simply adding one or more of theoligonucleotides of this invention to the original formulation.

Based on their immunostimulatory properties, one or more of theoligonucleotides of this invention can also be administered to a subjectin vivo to treat a tumoral disease or an immune system disorder.

Examples of common tumoral diseases are: Chronic Myelogenous Leukemia,Melanoma, Kaposi's Sarcoma, Multiple Myeloma, Renal Cell Carcinoma,Bladder Cancer, Lung Cancer, Skin Cancer, Brest Cancer, Colon Cancer andUterus Cancer. Examples of common immunological disorders are: Allergy,Severe Combined Immunodeficiency, Chronic Granulomatous disease, andAcquired Immunodeficiency Disease.

The pharmaceutical composition for these treatments may include one ormore of the oligonucleotides of this invention together with carriers,thickeners, diluents, buffers, preservatives, surface active agents,anti-microbial agents, anti-inflammatory agents, anesthetics and thelike. The formulation can be liquid or lyophilized.

The pharmaceutical composition may be administered in a number of waysdepending of whether local or systemic treatment is desired, and on thearea to be treated. Administration may be done topically, orally, byinhalation or parenterally. Formulation for topical administration mayinclude ointments, lotions, creams, gels, drops, suppositories, sprays,liquids and powders. Pharmaceutical carriers, aqueous, powder or oilybases, thickeners and the like may be necessary or desirable.Formulations for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,tablets and the like. Thickeners, flavorings, diluents, emulsifiers andthe like may be necessary or desirable. Formulations for parenteraladministration include sterile aqueous solutions, which may also containbuffers, diluents and other additives. Alternatively, one or more of theoligonucleotides of this invention can be contacted with immunocompetentcells (i.e. B cells or plasmacytoid dendritic cells) obtained from asubject having a tumoral disease or an immune system deficiency “exvivo” and activated cells can then be reintroduced in the subject.

EXAMPLE 1

Materials and Methods

The following materials and methods were used generally throughout theexamples.

1) Oligonucleotides

Oligonucleotides having phosphorothioate internucleotide linkages werepurchased, purified by high-pressure liquid chromatography (HPLC), fromOperon Technologies (Alameda, Calif.) or Annovis (Aston, Pa.) or OligosEtc (Bethel, Me.). Oligonucleotides were suspended in depyrogenatedwater and kept at −20° C. until used.

2) Antibodies

Antibodies used in assays were purchased from Serotec (Raleigh, N.C.).

3) Peripheric Blood Mononuclear Leukocytes (PBMC)

Blood was obtained by venipuncture from healthy donors using heparin asanticoagulant. PMBC were isolated by Ficoll-Hypaque (Sigma DiagnosticsInc., St. Louis, Mo.) density gradient centrifugation. Briefly, bloodsamples diluted 1:2 in RPMI-1640 medium (PAA laboratories GmbH, Linz,Austria) supplemented with 2.0 mM L-glutamine and 50.0 μg/ml gentamicinand 20 mM HEPES were centrifuged at 1000×g for 40 minutes at 20° C. PMBCwere isolated, washed and suspended in medium supplemented with 10%fetal calf serum.

4) Purification of Cells

B lymphocytes and plasmacytoid dendritic cells were purified from humanPMBC by positive selection using the MACS magnetic cell separationsystems (Miltenyi Biotec, Germany).

5) Cell Proliferation Assays

Blood was obtained by venipuncture from healthy donors using heparin asanticoagulant. PMBC were isolated by Ficoll-Hypaque (Sigma DiagnosticsInc., St. Louis, Mo.) density gradient centrifugation. Briefly, bloodsamples diluted 1:2 in RPMI-1640 medium (PAA laboratories GmbH, Linz,Austria) supplemented with 2.0 mM L-glutamine and 50.0 μg/ml gentamicinand 20 mM HEPES were centrifuged at 1000×g for 40 minutes at 20° C. PMBCwere isolated, washed and suspended in medium supplemented with 10%fetal calf serum.

6) IL6 Assay

PBMC (3×10⁵/well) were cultured as described above with ODNs (6 μg/ml)for 24 hr. After this, supernatants were collected and IL6 levelsmeasured by ELISA. Briefly, 96 well micro titer plates (NUNC, Denmark)were coated with anti-IL6 antibodies and blocked with RPMI 1640 mediasupplemented with 10% (v/v) heat inactivated FCS. IL6 was detectedcalorimetrically using biotin-labeled antibodies followed byperoxidase-conjugated strepto-avidin and then peroxidase-specificcalorimetric substrate. Standard curves were generated using knownamounts of recombinant IL6. The detection limit of these assays was 30pg/ml. All assays were performed in duplicate.

6) IgM Secretion Assay

PBMC (3×10⁵/well) were cultured as described above with ODNs (1.5 μg/ml)for 72 hr. After this, supernatants were collected and IgM assayed byELISA. Briefly, 96 microtiter plates (NUNC, Denmark) were coated withanti-lgM antibodies and blocked with RPMI 1640 media. IgM was detectedcalorimetrically using peroxidase-labeled antibodies followed byperoxidase-specific calorimetric substrate. Standard curves weregenerated using known amounts of purified IgM. The detection limit ofthese assays was 50 ng/ml. All assays were performed in duplicate.

6) Flow Cytometry

Staining of surface antigens was performed as described (J. Fló and E.Massouh. Age-related changes of native and memory CD4 rat lymphocytesubsets in mucosal and systemic lymphoid organs. Developmental andcomparative Immunology 21: 443-453, 1997). Anti CD19 (Clone LT19), CD86(Clone BU63), CD40 (clone LOB 7/6), CD4 (clone S 3.5), MHC class I(Clone W6/32) and MHC class II (Clone WR 18) antibodies were purchasedfrom Serotec (Raleigh, N.C., USA).

Flow cytometric data of 10,000 cells/sample were acquired on a FACScan(Becton Dickinson Immunocytometry Systems, San Jose, Calif.). Data wereanalyzed using the computer program Win MDI, 2.8, Interface FlowCytometry Application (Joseph Trotter Copyright 1993-1998).

7) Immunization of Monkeys Against Hepatitis B Surface Antigen (HBsAg)and Evaluation of the Humoral Response

Twelve monkeys of the species Cebus Apella (2.5-3.5 Kg) were immunizedwith a pediatric dose of AgB (Pablo Cassará, Buenos Aires, Argentina)containing 10 μg of HBsAg adsobed to alumina (25 mg of Al³⁺/mg ofHBsAg). This was administered alone (n=3, 1 female, 2 males) or combinedwith indicated phosphorothioate ODN (150 μg/dose) (n=3, 1 female, 2males). All vaccines were administered intramuscular (i.m.) in thequadriceps muscle in a total volume of 1 ml. Monkeys were maintained inthe animal facility of the CEMIC (Centro Medico de InvestigacionesClinicas), Buenos Aires, Argentina. Animals were monitored daily byspecialists and weighed once per week. Plasma was recovered byintravenous (i.v.) puncture before and at various times afterimmunization and immediately assayed for antibodies using the commercialkit AUSAB (Abbot Laboratories, Illinois, USA). Titers are expressed inmilliinternational units per ml.

EXAMPLE 2

Selection of Oligonucleotide Sequences

WO 96/02555 and U.S. Pat. No. 6,239,116 patents teach that to beimmunostimulatory, oligonucleotides require sequences containingunmethylated CpG motifs (WO 96/02555, col. 13 lines 19-20 and U.S. Pat.No. 6,239,116, col. 6 lines 1-3). EP 0 468 520 teaches that to beimmunostimulatory, oligonucleotides require a palindromic sequence of atleast 6 nucleotides long to be satisfactory (EP 0 468 520, col. 11,lines 34-37). Therefore, several non-CpG oliqonucleotides, withoutpalindromic sequences of at least 6 nucleotides long were probed usingproliferation assays, cell differentiation assays, cytokine IL6secretion assays and IgM secretion assays performed on human peripheralblood mononuclear leukocytes (PBMC). As a positive control, the CpGoligonucleotide 2006 of composition:

5′ TCGTCGTTTTGTCGTTTTGTCGTT 3′

and phosphorothioate bonds described by Hartman and Krieg (Hartmann, G.,Krieg, A. M. 2000. Mechanism and function of a newly identified CpG DNAmotif in human primary B cells. J. Immunol. 164:944.) was used. As abackground control the phosphorothioate oligonucleotide IMT 023 (SEQ IDNo 9) or IMT 022 (SEQ ID No 8) with very low activity on human cellswere used.

Also as a “presumably” negative control an oligonucleotide with the samecomposition of the oligonucleotide 2006 but in which all the CpGdinucleotides have been replaced by GpC dinucleotides:

5′ TGCTGCTTTTGTGCTTTTGTGCTT 3′

was used. This oligonucleotide was named ODN IMT 021 (SEQ ID No 1).

FIG. 1 shows a proliferation assay performed with the above describedoligonucleotides.

It was found, surprisingly, that the non CpG oligonucleotide IMT 021 wasas active as the 2006 CpG oligonucleotide in proliferation assays (FIG.1 and Table 1) if used at 1.5 mug/ml and approximately 40-60% active ifused at 0.375 mug/ml.

TABLE 1 Induction of peripheral white blood cell proliferation byPhosphorothioate CpG (2006) and non-CpG oligonucleotides (IMT 021)PROLIFERATION INDEX (ODN at 1.5 μg/ml) (ODN at 0.375 μg/ml) ODN (S)SEQUENCE (5′-3′) Avg. N SD Avg. N SD 2006 TCGTCGTTTTGTCGTTTTGTCGTT 17.954 2.39 13.83 14 1.55 IMT 021 TGCTGCTTTTGTGCTTTTGTGCTT 17.41 5 2.74 8.0215 1.21 MT 021 (SEQ. ID N^(O) 1) Avg.: Average; N: Number of Data; SD:Standard Deviation; ODN(S): phosphorothioate ODN

Immunostimulation was also evaluated by Flow Cytometry using the CD 19general marker for human B cells and the CD 86 activation marker forhuman B cells.

FIG. 2 shows the activation of human B cells incubated with the non CpGODN (S) IMT 021 as compared to the activation induced by incubation withthe CpG ODN (S) 2006. As can be observed, both ODNs show similaractivation pattern as compared with the ODN(S) IMT 023 backgroundcontrol.

IL6 secretion was also evaluated in supernatants of PBMC incubated withthe CpG ODN (S) 2006 and the non CpG ODN (S) IMT 021 (Table 2).

TABLE 2 Induction of human IL-6 secretion by phosphorothioate CpG andnon-CpG oligonucleotides 2006 and IMT 021 IL-6 (pg/ml) ODN (S) expt. 1expt. 2 expt. 3 expt. 4 cells alone 0 0 0 0 2006 568 159 597 374 IMT 021348 384 836 596 IMT 021 (SEQ. ID N^(O) 1)

Results of this assay also indicate that the non-CpG ODN (S) IMT 021 isan effective immunostimulant. Therefore, other non-CpG sequence variantsof the 2006 oligonucleotide were investigated. Table 3 shows the resultsfor six of these variants in which, the Cs or Gs of all the CpGs of thisODN were replaced by other nucleotides. As can be observed, the Gs ofthe CpGs in the ODN(S) 2006 are not necessary for B cell proliferationor IL6 secretion. However, modification of the Cs of the CpGs isdetrimental if the replacement is for As or Gs but not if it is for Ts.These results clearly indicate that stimulation of the B cellproliferation and IL6 secretion by the ODN(S) 2006 is not at allassociated with integrity of the CpG group.

TABLE 3 Induction of peripheral white blood cell proliferation and IL6secretion by non-CpG variants of the ODN(S) 2006 oligonucleotidePROLIFERATION INDEX IL-6 (pg/ml) (ODN at 0.375 μg/ml) (ODN at 6.0 μg/ml)ODN (S) SEQUENCE (5′-3′) Avg. N SD Avg. N SD 2006TCGTCGTTTTGTCGTTTTGTCGTT 13.83 14 1.55 215.8 4 83.4 IMT 504TCATCATTTTGTCATTTTGTCATT 13.08 2 3.80 285.2 2 39.5 IMT 505TCCTCCTTTTGTCCTTTTGTCCTT 12.98 2 4.73 218.9 2 3.1 IMT 506TCTTCTTTTTGTCTTTTTGTCTTT 11.66 4 2.77 228.9 4 161.0 IMT 501TAGTAGTTTTGTAGTTTTGTAGTT 5.42 2 2.03 66.8 2 54.0 IMT 502TGGTGGTTTTGTGGTTTTGTGGTT 7.16 2 3.24 89.7 2 91.4 IMT 503TTGTTGTTTTGTTGTTTTGTTGTT 9.87 2 1.90 334.9 2 215.5 IMT 504 (SEQ ID N^(O)2); IMT 505 (SEQ ID N^(O) 3); IMT 506 (SEQ ID N^(O) 4); IMT 501 (SEQ IDN^(O) 5); IMT 502 (SEQ ID N^(O) 6); IMT 503 (SEQ ID N^(O) 7)

EXAMPLE 3

Effect of Structure Modification on the Immunostimulatory Activity ofNon-CpG Oligonucleotides: Definition of the Active Motif.

In order to study the influence of the primary structure on theimmunostimulatory activity of the non-CpG ODNs, several variants of the2006 and IMT 021 oligonucleotides were synthesized. Table 4 shows theprimary structure of some of the IMT 021 variants and the results of aproliferation and IL-6 assays performed in order to evaluate itsimmunostimulatory activity.

The ODN(S) IMT 021 contains T in positions 7,8,9,10,12,15,16,17,18,20,23and 24. Replacement of these Ts with As (ODN IMT 022) or Cs (ODN IMT023) results in a very significant loss of activity (about 76% and 75%respectively) in the proliferation assay and also in the IL-6 secretionassay (84% and 88% respectively). These results indicate that some orall the Ts in positions 7,8,9,10,12,15,16,17,18,20,23 and 24 arecritical for the ODN IMT 021 immunostimulatory activity.

TABLE 4 Induction of peripheral white blood cell proliferation and IL-6secretion by Phosphorothioate non-CpG oligonucleotides derived from theinmunostimulatory IMT 021 oligonucleotide PROLIFERATION INDEX IL-6(pg/ml) (ODN at 0.375 μg/ml) (ODN at 6.0 μg/ml) ODN (S) SEQUENCE (5′-3′)Avg. N SD Avg. N SD IMT 021 TGCTGCTTTTGTGCTTTTGTGCTT 8.02 15 1.21 181.34 49.9 IMT 022 TGCTGCAAAAGAGCAAAAGAGCAA 1.24 2 0.30 0.0 2 0.0 IMT 023TGCTGCCCCCGCGCCCCCGCGCCC 1.07 4 0.12 0.0 3 0.0 IMT 021 (SEQ. ID N^(O)1); IMT 022 (SEQ. ID N^(O) 8); IMT 023 (SEQ. ID N^(O) 9)

The analysis of hundreds of ODNs (unshown) allowed definition of a coresequence (motif) responsible of the immunostimulatory activity asmeasured by B cell proliferation and IL6 secretion. This motif is thefollowing:X₁X₂X₃X₄X₅X₆X₇X₈,

wherein X₁ is C,T,G or A (preferably T or C);

wherein X₂ is C,T,G or A;

wherein X₇ is C,T,G or A (preferably G);

wherein at least three, and preferably all, of X₃, X₄, X₅, X₆ and X₈ areT.

Table 5 shows the effect of changes in each of the nucleotides of thetwo non-CpG motifs present in the ODN IMT 504 (motif: CATTTTGT).Replacement of the C in position 1 of the motif by A (ODN IMT 531) or G(ODN IMT 533) resulted in a loss of about 50% in the activity. However,replacement of this C by T (ODN IMT 532) does not change the activity.Thus, in order to obtain maximal activity the first position of themotif should be occupied by a pyrimidine nucleotide (C or T). Inposition 2, A (ODN IMT 504) or T (ODN IMT 535) or C (ODN IMT 534) areequivalent options.

In order to study the influence of a G nucleotide in position 2 of themotif without introduction of a CpG dinucleotide, ODNs with a T in thefirst position of the motif were synthesized (Table 6). As can beobserved, any nucleotide in the second position of the immunostimulatorymotif is equivalent.

Replacement of the G in position 7 of the immunostimulatory motif by A(ODN IMT 541), T (ODN IMT 542) or C (ODN IMT 543) is detrimental (Table5). Thus, in order to obtain maximal activity position 7 should bepreferably G.

Regarding to the positions of the immunostimulatory motif occupied byTs, the most sensitive (as measured by a change for an A) are 4 (ODN IMT538), 5 (ODN ITM 539) and 8 (ODN IMT 544) and less sensitive are 3 (ODNIMT 537) and 6 (ODN IMT 540). Replacement of two or more of the Tswithin the motif results in more than 70% loss of activity (ODNs IMT545, IMT 546, IMT 547, IMT 548, IMT 549, IMT 550, IMT 551 and IMT 552).Therefore, in order to obtain a significant immunostimulatory activitythree or more of positions 3,4,5,6 and 8 should be occupied by T.

TABLE 5 Induction of peripheral white blood cell proliferation and IL-6secretion by phosphorothioate non-CpG oligonucleotides of this inventionderived from ODN IMT 504 PROLIFERATION INDEX IL-6 (pg/ml) (ODN at 0.375μg/ml) (ODN at 6.0 μg/ml) ODN (S) SEQUENCE (5′-3′) Avg. N SD Avg. N SDIMT 022 TGCTGCAAAAGAGCAAAAGAGCAA 1.34 24 0.52 42.7 6 29.7 IMT 504TCATCATTTTGTCATTTTGTCATT 13.85 16 4.36 171.6 6 39.8 IMT 531TCATAATTTTGTAATTTTGTCATT 7.59 12 2.71 137.4 6 124.4 IMT 532TCATTATTTTGTTATTTTGTCATT 13.80 12 3.41 124.2 6 78.4 IMT 533TCATGATTTTGTGATTTTGTCATT 6.20 12 2.71 118.3 6 76.0 IMT 534TCATCCTTTTGTCCTTTTGTCATT 11.67 12 4.49 173.7 6 31.4 IMT 535TCATCTTTTTGTCTTTTTGTCATT 12.49 12 2.51 160.6 6 39.0 IMT 537TCATCAATTTGTCAATTTGTCATT 10.17 12 4.00 183.5 6 63.6 IMT 538TCATCATATTGTCATATTGTCATT 7.12 12 2.95 133.2 6 86.7 IMT 539TCATCATTATGTCATTATGTCATT 9.80 12 4.22 171.7 6 53.4 IMT 540TCATCATTTAGTCATTTAGTCATT 11.94 12 2.55 163.1 6 43.0 IMT 541TCATCATTTTATCATTTTATCATT 9.30 12 2.20 177.2 6 40.4 IMT 542TCATCATTTTTTCATTTTTTCATT 9.78 12 2.60 151.1 6 40.2 IMT 543TCATCATTTTCTCATTTTCTCATT 4.75 12 2.25 137.9 6 77.6 IMT 544TCATCATTTTGACATTTTGACATT 7.16 12 2.11 124.4 6 42.7 IMT 545TCATCATTTAGACATTTAGACATT 4.19 12 1.81 129.4 6 53.8 IMT 546TCATCATTATGACATTATGACATT 2.90 12 1.16 105.0 6 67.2 IMT 547TCATCATATTGACATATTGACATT 3.34 12 1.16 110.3 6 43.5 IMT 548TCATCATTAAGACATTAAGACATT 3.40 12 1.53 91.5 6 33.1 IMT 549TCATCATATAGACATATAGACATT 3.51 12 1.03 121.3 6 41.3 IMT 550TCATCATAATGACATAATGACATT 1.91 12 0.71 79.3 6 30.8 IMT 551TCATCATAAAGACATAAAGACATT 2.17 12 0.72 60.7 6 26.4 IMT 552TCATCAAAAAGACAAAAAGACATT 1.37 12 0.83 21.6 6 13.5 IMT 022 (SEQ. ID N^(O)8); IMT 504 (SEQ ID N^(O) 2); IMT 531 (SEQ ID N^(O) 14); IMT 532 (SEQ IDN^(O) 15); IMT 533 (SEQ ID N^(O) 16); IMT 533 (SEQ ID N^(O) 17); IMT 534(SEQ ID N^(O) 18); IMT 535 (SEQ ID N^(O) 19); IMT 537 (SEQ ID N^(O) 30);IMT 538 (SEQ ID N^(O) 31); IMT 539 (SEQ ID N^(O) 32); IMT 540 (SEQ IDN^(O) 33); IMT 541 (SEQ ID N^(O) 34); IMT 542 (SEQ ID N^(O) 35); IMT 543(SEQ ID N^(O) 36); IMT 544 (SEQ ID N^(O) 37); # IMT 545 (SEQ ID N^(O)38); IMT 546 (SEQ ID N^(O) 39); IMT 547 (SEQ ID N^(O) 40); IMT 548 (SEQID N^(O) 41); IMT 549 (SEQ ID N^(O) 42); IMT 550 (SEQ ID N^(O) 43); IMT551 (SEQ ID N^(O) 44); IMT 552 (SEQ ID N^(O) 45)

TABLE 6 Induction of peripheric white blood cell proliferation and IL-6secretion by Phosphorothioate non-CpG oligonucleotides with T in thefirst position of the immunostimulatory motif PROLIFERATION INDEX IL-6(pg/ml) (ODN at 0.375 μg/ml) (ODN at 6.0 μg/ml) ODN (S) SEQUENCE (5′-3′)Avg. N SD Avg. N SD IMT 532 TCATTATTTTGTTATTTTGTCATT 10.10 4 1.61 221.74 35.3 IMT 197 TCATTTTTTTGTTTTTTTGTCATT 10.84 4 1.06 386.1 4 123.3 IMT198 TCATTGTTTTGTTGTTTTGTCATT 10.00 4 1.75 235.3 4 27.1 IMT 199TCATTCTTTTGTTCTTTTGTCATT 12.36 3 1.71 235.0 4 6.0 IMT 532 (SEQ ID N^(O)15); IMT 197 (SEQ. ID N^(O) 10); IMT 198 (SEQ ID N^(O) 11); IMT 199 (SEQID N^(O) 12)

To investigate the effect of changes in the position of the motif withinthe ODN chain, the non-CpG motif CATTTTGT present in ODN IMT504 wasintroduced into different locations in a 24 nucleotides long poly Tchain (FIG. 3). As can be seen, the poly T ODN (S) by itself has asignificant mitogenic activity. On the other hand, introduction of onlyone CATTTTGT motif results in an increment of 1.2 to 1.8 times asmeasured by proliferation assays, or 2.4 to 3.5 times as measured byIL-6 secretion assays, in the immunostimulatory activity of the polyTchain depending on where the motif is located. A distance of at leasttwo nucleotides from the 5′ end or four from the 3′ end seems to benecessary in order to reach maximal activity (ODNs IMT174 to IMT 179).Introduction of two motifs in optimal positions (ODN IMT182) results inan increment of 1.9 times as measured by proliferation assays, or 2.9times as measured by IL-6 secretion assays, in the immunostimulatoryactivity of the polyT chain. This indicate that contribution of thesecond motif is negligible. On the other hand, the activity of the mosteffective ODNs like ODN IMT504 is more than 3 times larger than theactivity of the poly T as measured by proliferation assays a fact thatsuggest that there is also a significant influence of the composition ofat least some of the nucleotides surrounding the motif of thisinvention, in the overall activity of the ODN.

EXAMPLE 4

Effect of Structure Modification on the Immunostimulatory Activity ofNon-CpG Oligonucleotides of this Invention: Influence of the NucleotideComposition Outside of the Active Motif.

Results shown in FIG. 3 indicate that composition of the oligonucleotideoutside the non-CpG core motif is important in order to reach optimalimmunostimulatory activity. Therefore, a number of oligonucleotides weresynthesized with changes in the composition of the nucleotidessurrounding the motifs. Table 7 shows the effect of changes in thecomposition of the first four nucleotides of the 5′ end and of the lastfour nucleotides the 3′ end in the immunostimulatory activity of ODN IMT504. As can be observed, the best choices are: C or G in position −1respect to the two motifs, C, T or G in position −2, any nucleotide inpositions −3 and −4, A or T in position +1, G in position +2, anynucleotide in positions +3 and G is the best choice in position +4 .

Of course, other nucleotide combinations not represented in this tablemay have equal or even better effect on the activity of the non CpGimmunostimulatory oligonucleotides. One of ordinary skill in the art canempirically determine other effective combinations.

TABLE 7 Induction of peripheric white blood cell proliferation bynon-CpG ODN(S) IMT 504 with variations in the composition outside thetwo immunostimulatory motifs PROLIFERATION INDEX IL-6 (pg/ml) (ODN at0.375 μg/ml) (ODN at 6.0 μg/ml) ODN (S) SEQUENCE (5′-3′) Avg. N SD Avg.N SD IMT 552 TGCTGCAAAAGAGCAAAAGAGCAA 1.9 12 0.7 <39.9 4 — IMT 504TCATCATTTTGTCATTTTGTCATT 11.0 8 0.9 171 2 14 IMT 559ACATCATTTTGTCATTTTGTCATT 13.4 4 1.0 128 3 3 IMT 560CCATCATTTTGTCATTTTGTCATT 12.1 4 0.9 145 4 32 IMT 561GCATCATTTTGTCATTTTGTCATT 9.5 4 1.1 209 4 74 IMT 562TAATCATTTTGTCATTTTGTCATT 12.4 4 1.5 171 4 24 IMT 563TTATCATTTTGTCATTTTGTCATT 11.1 3 0.7 172 4 63 IMT 564TGATCATTTTGTCATTTTGTCATT 12.8 4 0.9 118 4 38 IMT 565TCCTCATTTTGTCATTTTGTCATT 14.1 4 0.5 135 4 25 IMT 566TCTTCATTTTGTCATTTTGTCATT 13.9 4 2.2 157 4 28 IMT 567TCGTCATTTTGTCATTTTGTCATT 12.9 4 0.7 259 4 25 IMT 568TCAACATTTTGTCATTTTGTCATT 10.3 4 1.1 153 4 35 IMT 569TCACCATTTTGTCATTTTGTCATT 15.0 4 1.2 199 3 12 IMT 570TCAGCATTTTGTCATTTTGTCATT 12.5 4 0.4 181 3 2 IMT 571TCATCATTTTGTCATTTTGTAATT 14.2 4 0.7 163 4 26 IMT 572TCATCATTTTGTCATTTTGTTATT 13.2 4 1.4 182 3 67 IMT 573TCATCATTTTGTCATTTTGTGATT 8.7 4 0.7 177 4 29 IMT 574TCATCATTTTGTCATTTTGTCCTT 8.1 4 0.7 179 3 58 IMT 575TCATCATTTTGTCATTTTGTCTTT 10.8 4 0.8 150 3 47 IMT 576TCATCATTTTGTCATTTTGTCGTT 12.6 4 1.1 202 4 55 IMT 577TCATCATTTTGTCATTTTGTCAAT 10.0 4 1.4 243 3 8 IMT 578TCATCATTTTGTCATTTTGTCACT 10.9 4 0.9 213 2 9 IMT 579TCATCATTTTGTCATTTTGTCAGT 10.7 4 1.7 182 4 18 IMT 580TCATCATTTTGTCATTTTGTCATA 11.7 4 1.4 194 4 36 IMT 581TCATCATTTTGTCATTTTGTCATC 10.7 4 1.0 173 4 18 IMT 582TCATCATTTTGTCATTTTGTCATG 12.1 4 0.8 242 3 231 IMT 552 (SEQ. ID N^(O)45); IMT 504 (SEQ ID N^(O) 2); IMT 559 (SEQ ID N^(O) 46); IMT 560 (SEQ.ID N^(O) 47); IMT 561 (SEQ ID N^(O) 48); IMT 562 (SEQ ID N^(O) 49); IMT563 (SEQ ID N^(O) 50); IMT 564 (SEQ ID N^(O) 51); IMT 565 (SEQ ID N^(O)52); IMT 566 (SEQ ID N^(O) 53); IMT 567 (SEQ ID N^(O) 54); IMT 568 (SEQID N^(O) 55); IMT 569 (SEQ ID N^(O) 56); IMT 570 (SEQ ID N^(O) 57); IMT571 (SEQ ID N^(O) 58); IMT 572 (SEQ ID N^(O) 59); # IMT 573 (SEQ IDN^(O) 60); IMT 574 (SEQ ID N^(O) 61); IMT 575 (SEQ ID N^(O) 62); IMT 576(SEQ ID N^(O) 63); IMT 577 (SEQ ID N^(O) 64); IMT 578 (SEQ ID N^(O) 65);IMT 578 (SEQ ID N^(O) 66); IMT 580 (SEQ ID N^(O) 67); IMT 581 (SEQ IDN^(O) 68); IMT 582 (SEQ ID N^(O) 69)

EXAMPLE 5

Effect of Structure Modification on the Immunostimulatory Activity ofNon-CpG Oligonucleotides of this Invention: Influence of the Size of theOligonucleotide

Table 8 shows that ODNs with one immunostimulatory motif are active ifthe chain is 16 or more nucleotides long. Activity is maximal if the ODNis 20 or more nucleotides long.

TABLE 8 Effect of the phosphorothioate non-CpG oligonucleotides size onB cells proliferation and IL6 secretion PROLIFERATION INDEX IL-6 (pg/ml)(ODN at 0.375 μg/ml) (ODN at 6.0 μg/ml) ODN (S) SEQUENCE (5′-3′) Avg. NSD Avg. N SD IMT 187 TTTTCATTTTGT 0.56 8 0.20 <50 4 — IMT 188TTTTCATTTTGTTTTT 2.76 8 1.73 147 4 40 IMT 189 TTTTCATTTTGTTTTTTTTT 8.218 3.19 227 4 21 IMT 175 TTTTCATTTTGTTTTTTTTTTTTT 8.92 8 1.80 220 4 18IMT 179 TTTTTTTTTTTTCATTTTGTTTTT 6.06 8 2.00 224 4 82 IMT 191TTTTCATTTTGTTTTTTTTTTTTTTTTT 6.64 8 2.37 205 4 15 IMT 187 (SEQ ID N^(O)70); IMT 188 (SEQ ID N^(O) 71); IMT 189 (SEQ ID N^(O) 72); IMT 179 (SEQID N^(O) 73); IMT 191 (SEQ ID N^(O) 77)

EXAMPLE 6

Induction of Peripheric White Blood Cell IgM Secretion byPhosphorothioate Non-CpG Oligonucleotides of this Invention

Induction of IgM secretion in peripheral white blood cells is anotherimportant marker of immunostimulatory activity of oligonucleotides.Table 9 shows the stimulation of IgM secretion by several of thephosphorothioate non-CpG oligonucleotides described above. As can beobserved, the most active non-CpG ODN(S)s in induction of proliferationand IL6 secretion are also the best in induction of IgM secretion.

TABLE 9 Induction of peripheral white blood cell IgM secretion byphosphorothioate non-CpG oligonucleotides of this invention IgMSECRETION (ng/ml) (ODN at 1.50 μg/ml) ODN (S) SEQUENCE (5′-3′) Avg. N SDcells alone 76 9 50 2006 TCGTCGTTTTGTCGTTTTGTCGTT 778 47 204 IMT 021TGCTGCTTTTGTGCTTTTGTGCTT 850 25 264 IMT 501 TAGTAGTTTTGTAGTTTTGTAGTT 58525 179 IMT 502 TGGTGGTTTTGTGGTTTTGTGGTT 555 19 277 IMT 503TTGTTGTTTTGTTGTTTTGTTGTT 640 19 220 IMT 504 TCATCATTTTGTCATTTTGTCATT 91224 244 IMT 505 TCCTCCTTTTGTCCTTTTGTCCTT 664 23 336 IMT 506TCTTCTTTTTGTCTTTTTGTCTTT 751 43 237 IMT 509 AAAAAACTAAAAAAAACTAAAAAA 19517 77 IMT 021 (SEQ. ID N^(O) 1); IMT 504 (SEQ ID N^(O) 2); IMT 505 (SEQID N^(O) 3); IMT 506 (SEQ ID N^(O) 4); IMT 501 (SEQ ID N^(O) 5); IMT 502(SEQ ID N^(O) 6); IMT 503 (SEQ ID N^(O) 7); IMT 509 (SEQ ID N^(O) 13)

EXAMPLE 7

Stimulation of the Expression of CD40, MHC I and MHC II in B Lymphocytesby Phosphorothioate Non-CpG Oligonucleotides of this Invention

As previously shown (FIG. 2), the non CpG ODN IMT 021 is able tostimulate the expression of CD86 on CD19+ cells (B lymphocytes). Inorder to extent this observation to other important cell surfacemarkers, human PMBC were incubated with ODN IMT 504, ODN 2006 as apositive control and ODN IMT 022 as a negative control (FIG. 4). As canbe observed, ODN IMT 504 is as active as the CpG ODN 2006 forstimulation of the expression of CD40, MHC I and MHC II on Blymphocytes.

EXAMPLE 8

Stimulation of Purified B Lymphocytes by Non-CpG Oligonucleotides ofthis Invention

Human CD19⁺ (B) cells were purified to more than 95% purity. Table 10and FIG. 5 shows that the immunostimulatory activity on this purifiedcells is comparable to the one observed using human PMBC. These resultsindicate that stimulation by the non-CpG oligonucleotides of thisinvention on human cells is direct.

TABLE 10 Induction of proliferation, IL6 and IgM secretion on purified Bcells by phosphorothioate non-CpG oligonucleotides of this inventionProliferation Index IL-6 (pg/ml) IgM (ng/ml) (ODN at 0.375 μg/ml) (ODNat 6.0 μg/ml) (ODN at 1.5 μg/ml) ODN (S) SEQUENCE (5′-3′) Ave. N SD Ave.N SD Ave. N SD IMT 022 TGCTGCAAAAGAGCAAAAGAGCAA 1.28 16 0.80 1019 5 665469 7 182 IMT 021 TGCTGCTTTTGTGCTTTTGTGCTT 24.48 16 5.16 9588 5 729 9469 386 IMT 504 TCATCATTTTGTCATTTTGTCATT 47.95 16 6.41 11002 5 884 1274 9252 IMT 506 TCTTCTTTTTGTCTTTTTGTCTTT 25.58 16 7.84 10933 3 1628 1083 10430 2006 TCGTCGTTTTGTCGTTTTGTCGTT 60.82 24 9.79 7631 6 997 1292 12 245IMT 021 (SEQ. ID N^(O) 1); IMT 022 (SEQ. ID N^(O) 8); IMT 504 (SEQ IDN^(O) 2); IMT 506 (SEQ ID N^(O) 4)

EXAMPLE 9

Immunostimulation by Phosphodiester Non-CpG Oligonucleotides of thisInvention

FIG. 6 and Table 11 shows the effect of phosphodiester non-CpGoligonucleotides of this invention on human PMBC. Since phosphodiesteroligonucleotides are very sensitive to nucleases they were added to theculture three times (0, 4, and 16 hs) to a final concentration of 30μg/ml. A very potent phosphodiester CpG ODN (ODN 2080) was used aspositive control in cytometric assays (Hartmann G, Weeratna R, Ballas ZK, Payette P, Suparto, I, Rasmussen W L, Wadschmidt M, Sajuthi D,Purcells R H, Davis H L, Krieg A M. Delineation of a CpGphosphorothioate oligodeoxynucleotide for activating primate immuneresponses in vitro and in vivo. J. Immunol.164, 1617-1624, (2000)). Ascan be observed, under these conditions phosphodiester ODNs bearing nonCpG motifs of this invention have immunostimulatory activity.

TABLE 11 Immune-stimulation by phosphodiester non-CpG oligonucleotidesof this invention IL-6 Secretion (ng/ml) (ODN at 6.0 μg/ml) ODN (S)SEQUENCE (5′-3′) Avg. N SD IMT 022 TGCTGCAAAAGAGCAAAAGAGCAA 23 8 10 IMT053 TTTTTTTTTTTTTTTTTTTTTTTT 24 4 24 IMT 504 TCATCATTTTGTCATTTTGTCATT207 6 46 IMT 506 TCTTCTTTTTGTCTTTTTGTCTTT 403 3 220 2006TCGTCGTTTTGTCGTTTTGTCGTT 228 4 40 IMT 022 (SEQ. ID N^(O) 8); IMT 504(SEQ ID N^(O) 2); IMT 053 (SEQ ID N^(O) 19); IMT 506 (SEQ ID N^(O) 4)

EXAMPLE 10

Induction of Cell Proliferation in Peripheral White Blood Cells ofMonkeys of the Species Cebus apella and Macaca fascicularis by Non-CpGODN(S)s of this Invention

Non-CpG ODN(S)s of this invention are not very effectiveimmunostimulants in mouse, pig and sheep. However, they are effective inmonkeys. For example, FIG. 7 shows the immunostimulatory activity ofnon-CpG ODN(S)s on peripheral white blood cells of monkeys of thespecies Cebus apella and Macaca fascicularis. According to these resultsthe most effective non-CpG ODNs in primates are those bearing theCATTTTGT motif. Therefore, monkeys can be used as animal models forresearch on clinical applications of these non-CpG ODN(S)s.

EXAMPLE 11

Vaccination of Subjects

Vaccine formulations containing the non-CpG ODNs adjuvant of thisinvention can be used to vaccinate subjects against a variety ofbacterial and viral disease agents and against tumoral cells. Table 12shows the effect of inoculation of monkeys of the species Cebus apellawith a vaccine formulation that includes recombinant Hepatitis B surfaceantigen (rHBsAg) and alumina in presence or absence of ODN IMT 504 orODN IMT 021 as adjuvant.

TABLE 12 Anti-HBs responses in Cebus apella immunized against HBsAg withIMT ODNs Post - prime Post - boost ODN (S) Pre - prime 14 days 28 days14 days None 0 0.4 ± 0.1 14 ± 20 190 ± 52  IMT 021 0 23 ± 32 231 ± 287902 ± 169 IMT 504 0 10 ± 14 113 ± 114 659 ± 296 2006 0 22 ± 24 67 ± 57705 ± 315

As can be observed there is a dramatic increment in the title ofanti-HBsAg in animals vaccinated with HBsAg plus IMT ODN(S) 504 or 21 ascompared with those vaccinated with HBsAg alone. CpG ODN 2006 have aperformance as adjuvant similar to the non-CpG ODNs of this invention.

In particular, a human can be vaccinated against hepatitis B byadministration of a vaccine formulation that includes recombinantHepatitis B surface antigen (rHBsAg) and alumina and one or more of theoligonucleotides of this invention as adjuvants. The amount of rHBsAg ineach dose, and the administration schedule, can vary as appropriate forthe age of the human. For example, for humans from about birth to about12 years old, a three dose schedule of from about 2.5 μg to about 5 μgof rHBsAg can be administered at 0, 1-3 months afterward and 4-18 monthsafterward, preferably at 0, 2 and 6 months. One or more of theoligonucleotides of this invention can be present in the formulationfrom about 10 μg to about 1,000 μg per dose.

For humans from about 12 to about 60 years old, a three dose schedule offrom about 5 μg to about 40 μg of rHBsAg can be administered at 0, 1-3months afterward and 4-18 months afterward, preferably at 0, 2 and 6months. One or more of the oligonucleotides of this invention can bepresent in the formulation from about 10 μg to about 1,000 μg per dose.

EXAMPLE 12

Stimulation of the Expression of Costimulatory Molecules in MalignantB-Lymphocytes by Phosphorothioate Non-CpG Oligonucleotides

The stimulation of malignant B lymphocytes recovered from blood ofpatients suffering chronic lymphocytic leukemia (CLL) byphosphorothioate non-CpG oligonucleotides of this invention was examinedby flow cytometric (FACS) analysis. The results of a typical FACSanalysis are shown in FIG. 8.

As can be seen the phosphorothioate non-CpG oligonucleotides IMT 021 andIMT 504 of this invention are able to stimulate expression of CD86, CD40and MHC class I surface costimulatory molecules on malignant Blymphocytes as well as the CpG ODN 2006.

EXAMPLE 13

Stimulation of the Expression of Costimulatory Molecules on PlasmacytoidDendritic Cells by Phosphorothioate Non-CpG Oligonucleotides of thisInvention

The stimulation of plasmacytoid dendritic cells recovered from blood ofnormal donors by phosphorothioate non-CpG oligonucleotides of thisinvention was examined by flow cytometric (FACS) analysis. The resultsof a typical FACS analysis are shown in FIG. 9. As can be seen thephosphorothioate non-CpG oligonucleotides IMT 021 and IMT 504 of thisinvention are able to stimulate expression of CD86, CD40 and MHC class Isurface molecules on plasmacytoid dendritic cells as well as the CpG ODN2006.

EXAMPLE 14

Treatment of Subjects with Tumoral Disease

Pharmaceutical formulations containing one or more of theoligonucleotides immunostimulatory of this invention, can be used totreat subjects against a variety of tumoral diseases. In particular, ahuman with a Melanoma can be treated by administration of apharmaceutical formulation containing the oligonucleotide of thisinvention as the active component. The amount of oligonucleotide in eachdose, and the administration schedule, can vary appropriate for thecorporal mass of the subject and stage in tumor progression. Forexample, for a human of about 70 Kg. having an advance, unresectablemetastatic melanoma, a dose of about 1 mg of the oligonucleotide of thisinvention can be administered 3 times per week during about 10 weeks.

1. An immunostimulatory oligonucleotide having 24 to 100 nucleotides,comprising the nucleotide sequence TCATCATTTTGTCATTTTGTCATT (SEQ ID No2).
 2. The immunostimulatory oligonucleotide of claim 1, wherein theimmunostimulatory oligonucleotide is encapsulated in a slow releasedelivery vehicle.
 3. The immunostimulatory oligonucleotide of claim 1,wherein the immunostimulatory oligonucleotide is included in apharmaceutically acceptable carrier.
 4. A pharmaceutical compositioncomprising an immunostimulatory oligonucleotide according to claim 1 anda pharmaceutically acceptable carrier.
 5. A composition according toclaim 4, wherein said immunostimulatory oligonucleotide is included in aplasmid.
 6. A composition according to claim 4, further comprising anantigen.
 7. A composition according to claim 6, wherein the antigen isselected from the group consisting of viruses, bacteria, fungi,parasites, tumor cells, toxins, allergens, proteins, glycolipids, andpolysaccharides.
 8. A composition according to claim 6, wherein theantigen is a viral antigen, a bacterial antigen, a human or animal tumorcell antigen, or a fungal antigen.
 9. A composition according to claim4, further comprising a plasmid encoding an antigen.
 10. A compositionaccording to claim 9, wherein the encoded antigen is a viral antigen, abacterial antigen, a human or animal tumor cell antigen, or a fungalantigen.
 11. An immunostimulatory oligonucleotide consisting of thenucleotide sequence TCATCATTTTGTCATTTTGTCATT (SEQ ID No 2).